Development, validation and application of LC–MS/MS method for quantification of amino acids, kynurenine and serotonin in human plasma

Dávid Virág, Márton Király, László Drahos, Andrea Edit Édes, Kinga Gecse, György Bagdy, Gabriella Juhász, István Antal, Imre Klebovich, Borbála Dalmadi Kiss, Krisztina Ludányi

Research output: Contribution to journalArticle

Abstract

Altered serotonergic neurotransmission is a key factor in several neurologic and psychiatric disorders such as migraine. Human and animal studies suggest that chronically low interictal serotonin levels of plasma and brain may facilitate increased activity of the trigeminovascular pathway, and may contribute to development of repeated migraine attacks. However, brain serotonin synthesis is affected by the concentration of tryptophan, its metabolites and a number of amino acids. In this work a simple and robust LC–MS/MS method for the quantitative determination of valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serotonin and kynurenine in human plasma has been developed and validated. Sample preparation was achieved by protein precipitation, using trifluoroacetic acid. Chromatographic separation was carried out on a Supelco Ascentis® Express C18 column (3.0 mm i.d. × 150 mm, 2.7 μm) equipped with an Agilent Zorbax Eclipse XDB C8 guard-column under isocratic conditions at a flow rate of 0.4 mL/min, over a 6.5 min run time. Mobile phase was 0.2% trifluoroacetic acid – acetonitrile (85:15, v/v). The eight analytes and two internal standards were ionized by positive electrospray ionization and detected in multiple reaction monitoring mode. A “fit-for-purpose” validation approach was adopted using surrogate matrix for the preparation of calibration samples. The calibration curves of all analytes showed excellent linearities with a correlation coefficient (r2) of 0.998 or better. Spiked surrogate matrix samples and pooled human plasma were used as quality control samples. Intra-day and inter-day precisions were less than 11.8% and 14.3%, and accuracies were within the ranges of 87.4–114.3% and 87.7–113.3%, respectively. Stability of the components in standard solutions, surrogate matrix, pooled plasma and processed samples were found to be acceptable under all relevant conditions. No significant carryover effect was observed. The surrogate matrix behaved parallel to human plasma when assessed by standard addition method and diluting the authentic matrix with surrogate matrix. The method was successfully applied for analysis of 800 human plasma samples to support a clinical study.

Original languageEnglish
Article number113018
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume180
DOIs
Publication statusPublished - Feb 20 2020

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Plasma (human)
Kynurenine
Serotonin
Amino Acids
Trifluoroacetic Acid
Tryptophan
Brain
Calibration
Migraine Disorders
Plasmas
Electrospray ionization
Isoleucine
Valine
Metabolites
Phenylalanine
Leucine
Quality control
Tyrosine
Animals
Flow rate

Keywords

  • Amino acid
  • Fit-for-purpose validation
  • Kynurenine
  • LC–MS/MS
  • Serotonin
  • Surrogate matrix

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

Cite this

Development, validation and application of LC–MS/MS method for quantification of amino acids, kynurenine and serotonin in human plasma. / Virág, Dávid; Király, Márton; Drahos, László; Édes, Andrea Edit; Gecse, Kinga; Bagdy, György; Juhász, Gabriella; Antal, István; Klebovich, Imre; Dalmadi Kiss, Borbála; Ludányi, Krisztina.

In: Journal of Pharmaceutical and Biomedical Analysis, Vol. 180, 113018, 20.02.2020.

Research output: Contribution to journalArticle

Virág, Dávid ; Király, Márton ; Drahos, László ; Édes, Andrea Edit ; Gecse, Kinga ; Bagdy, György ; Juhász, Gabriella ; Antal, István ; Klebovich, Imre ; Dalmadi Kiss, Borbála ; Ludányi, Krisztina. / Development, validation and application of LC–MS/MS method for quantification of amino acids, kynurenine and serotonin in human plasma. In: Journal of Pharmaceutical and Biomedical Analysis. 2020 ; Vol. 180.
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AU - Juhász, Gabriella

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N2 - Altered serotonergic neurotransmission is a key factor in several neurologic and psychiatric disorders such as migraine. Human and animal studies suggest that chronically low interictal serotonin levels of plasma and brain may facilitate increased activity of the trigeminovascular pathway, and may contribute to development of repeated migraine attacks. However, brain serotonin synthesis is affected by the concentration of tryptophan, its metabolites and a number of amino acids. In this work a simple and robust LC–MS/MS method for the quantitative determination of valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serotonin and kynurenine in human plasma has been developed and validated. Sample preparation was achieved by protein precipitation, using trifluoroacetic acid. Chromatographic separation was carried out on a Supelco Ascentis® Express C18 column (3.0 mm i.d. × 150 mm, 2.7 μm) equipped with an Agilent Zorbax Eclipse XDB C8 guard-column under isocratic conditions at a flow rate of 0.4 mL/min, over a 6.5 min run time. Mobile phase was 0.2% trifluoroacetic acid – acetonitrile (85:15, v/v). The eight analytes and two internal standards were ionized by positive electrospray ionization and detected in multiple reaction monitoring mode. A “fit-for-purpose” validation approach was adopted using surrogate matrix for the preparation of calibration samples. The calibration curves of all analytes showed excellent linearities with a correlation coefficient (r2) of 0.998 or better. Spiked surrogate matrix samples and pooled human plasma were used as quality control samples. Intra-day and inter-day precisions were less than 11.8% and 14.3%, and accuracies were within the ranges of 87.4–114.3% and 87.7–113.3%, respectively. Stability of the components in standard solutions, surrogate matrix, pooled plasma and processed samples were found to be acceptable under all relevant conditions. No significant carryover effect was observed. The surrogate matrix behaved parallel to human plasma when assessed by standard addition method and diluting the authentic matrix with surrogate matrix. The method was successfully applied for analysis of 800 human plasma samples to support a clinical study.

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